The use of nucleic acid probes to detect particular target nucleic acid sequences in samples containing at least one nucleic acid is of vast utility to research, medicine and forensics. Because nucleic acid probes are highly specific for their target sequences, they can be used as diagnostic reagents to detect the presence of a particular nucleic acid, as well as features within that nucleic acid. Commercial nucleic acid probe assays are being developed for the detection of infectious microorganisms, viruses, mutations in the human genome, as well as for fingerprinting human and other species' genomes. Research applications of nucleic acid probes are many, having been extensively utilized in recombinant DNA work for over 10 years.
Nucleic acid probe hybridization involves the detection of a target nucleic acid (e.g., RNA or DNA), either bound to a solid support or free in solution, using a labeled complementary probe nucleic acid or analog thereof (e.g., peptide nucleic acids (PNAs), methylene methyl amino oligonucleotides, and other polymers having Watson-Crick bases). Nucleic acid probe assays fall into two general categories, i.e., free-solution (or homogeneous) assays and solid support (or heterogeneous) assays.
In homogeneous assays, the target and probe nucleic acids are dissolved in solution. Target nucleic acid is first extracted from the sample, typically denatured to convert it to single-stranded form, and dissolved in hybridization buffer. Extraction of target nucleic acid from the sample and denaturation thereof can be accomplished by the procedure disclosed by Maniatis et al. in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory (1982), pages 191 to 198. A labeled probe complementary to the target nucleic acid is added to this solution and allowed to hybridize with the target sequence. When the hybridization reaction is complete, a suspension of hydroxyapatite (calcium hydroxide) is added. The hydroxyapatite selectively binds double-stranded probe/target nucleic acid duplexes as well as other double-stranded molecules, but does not bind unannealed single-stranded molecules. The insoluble hydroxyapatite with probe/target sequence duplex bound thereto is separated from the hybridization medium by centrifugation and washed to remove traces of unreacted probe molecules. If the probe has an isotopic label, the amount of probe bound to the hydroxyapatite is quantitated by scintillation counting. Other conventional means can be used to detect and quantitate nonisotopically labeled probe bound to the hydroxyapatite.
Heterogeneous assays can be performed in many ways. A particularly common method comprises binding a single-stranded target nucleic acid to a nitrocellulose or nylon filter in an irreversible manner. This can be accomplished by applying the target nucleic acid to the filter, and then baking them at temperatures of 70.degree. C. to 80.degree. C. for about one to about two hours under reduced pressure, i.e., at a pressure of less than 1 psi. The filter with sample nucleic acid bound thereto is subsequently prehybridized by immersion in an aqueous solution containing salts, protein, nonreactive DNA or RNA, sodium dodecyl sulfate detergent, buffer, EDTA, and formamide to block nonspecific binding sites on its surface. See, e.g., Grunstein et al, "Colony Hybridization: A Method for the Isolation of Cloned DNAs that Contain a Specific Gene," Proc. Nat'l Acad. Sci., 72(10):3961-3965, 1975.
When the prehybridization step is complete, labeled probe is dissolved in an aqueous solvent and is added to the solution containing the prehybridized filter to which the sample nucleic acid is bound. The probe is allowed to hybridize with the filter-bound sample nucleic acid until formation of sample/probe duplexes has gone to completion. The filter is then removed from the hybridization solution and washed with a buffered salt solution at a controlled temperature to remove nonspecifically bound labeled probe sequences. After the washing step, only labeled probe molecules which are specifically annealed to matching sample target sequences remain on the filter. The washed filter can be autoradiographed, or other appropriate conventional means can be used to detect the label and determine the amount and location of the bound probe, and thereby the location of the complementary sample sequences originally applied. See, e.g., Meinkoth et al., "Hybridization of Nucleic Acids Immobilized on Solid Supports," Analytical Biochemistry, 138:267, 1984; and Thomas, "Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose," Proc. Nat'l Acad. Sci., 77(9):5201-5205, 1980.
All nucleic acid probe assays require a step in which a labeled probe nucleic acid is hybridized to a target nucleic acid sequence. The time required for such hybridization is often a critically limiting factor in nucleic acid probe assays. The rate of hybridization is affected by several factors, such as ionic strength, temperature, concentration of the reactant molecules, and the presence of denaturing solvent. Concentration of the reactant molecules is perhaps the most important of these factors, because it limits the rate at which the random collisions between the complementary nucleic acid probe and target sequences occur as required to bring about hybridization. Once two complementary nucleic acid molecules have appropriately collided, they rapidly hybridize to form a thermodynamically stable duplex that does not spontaneously dissociate into its single-stranded components.
It has been found that the rate of DNA or RNA hybridizaton in homogeneous assays can be accelerated by the addition to the hybridization medium of water soluble polymers, such as dextran sulfate, polyvinyl pyrrolidone, or tetraethyl ammonium chloride. See, e.g., Wetnur, Biopolymers, 14:2517-2524, 1975; Chang et al., Biopolymers, 13:1847-1858, 1975; and Kohne, ACPR:20-29, November 1986. The mechanism by which these polymers enhance the rate of hybridization of DNA or RNA molecules is believed to involve a reduction in the effective solvent volume available to the nucleic acids in solution. The negatively charged polymers complex with available solvent molecules from around the nucleic acid molecules, resulting in an effective increase in concentration of DNA or RNA molecules relative to each other. Such concentration is believed to be effective to increase the number of collisions between complementary sequences, and to thereby produce faster hybridization rates.
Such rate-enhancing compounds have been found to increase nucleic acid probe hybridization rates by 10 to 200 fold in homogeneous assays, thereby making possible hybridization times of 1 to 2 hours, rather than overnight. In the case of short synthetic nucleic acid probes, hybridization reactions can be completed in less than 15 minutes if high concentrations of oligomeric probe, for example 1 milligram per milliliter, are used along with rate-enhancing compounds. In general, however, the hybridization reaction for nucleic acid probe assays requires 1 to 2 hours when probes of 100 or more nucleotides in length are used in homogeneous hybridization assays. Moreover, rate-enhancing compounds have not been found to significantly enhance the hybridization rate for heterogeneous assays.
In order to supply the frequent need of researchers and others to collect a dense amount of nucleic acid molecules, for example on a carrier membrane, instruments are available commercially which can separate nucleic acid from a gel or can isolate or concentrate nucleic acid molecules from a solution thereof. The operation of such electroelution or electrophoretic concentration devices takes advantage of the fact that, due to the presence of phosphate groups on the nucleic acid backbone, DNA and RNA in aqueous solution are highly negatively charged molecules. When a voltage is applied across platinum wire electrodes placed in a solution of RNA or DNA, the resulting current flow through the solution causes the negatively charged nucleic acid molecules to migrate toward the positive electrode (anode) and concentrate on its surface.
In the aforementioned commercial devices, this principle is used to electrophoretically concentrate the migrating DNA or RNA molecules from a solution, or from agarose or acrylamide containing such molecules, onto the surface of a liquid permeable, for example a cellulose, collector membrane which is impermeable to the nucleic acid molecules and is positioned to prevent such molecules from contacting the anode. Usually, devices of this sort are configured with two chambers separated by the membrane. In one chamber the gel or nucleic acid-containing moiety is placed in a buffered solution near but not against one side of the membrane. The second chamber contains only buffered solution in contact with the other side of the membrane so that aqueous solution contacts both sides of the latter. Platinum wire electrodes present in the respective chambers are connected to a constant direct voltage power supply, the electrode in the chamber containing the nucleic acid to be concentrated being connected to the negative terminal of the source to provide a cathode, and the other electrode being connected to the positive terminal thereof to provide an anode. The electric potential impressed across the electrodes by the source, causes current flow through the aqueous solutions and is effective to cause the negatively charged nucleic acid molecules in the cathode chamber electrophoretically to migrate toward and be concentrated onto the side of the membrane or disc exposed in the cathode chamber. The nucleic acid becomes deposited on the membrane or disc during the procedure. Upon completion of the concentration step, the electrodes are disconnected from the source, and the nucleic acid deposited on the membrane can be easily removed therefrom, as by washing.
Depending upon the type of membrane used therein, the commercial devices can also be used to bind to the membrane the nucleic acids concentrated thereon. For example, when a membrane of modified nylon is used, the nucleic acids concentrated thereon are bound thereto upon contact. On the other hand, when a membrane of nitrocellulose is used, the nucleic acids concentrated thereon can be bound thereto upon removal of the membrane from the instrument. Such binding can be accomplished by baking at a temperature of 70.degree. C. for about one to about two hours at reduced pressure, i.e. less than 1 psi.
Examples of commercial electrophoretic concentration/elution instruments of the type discussed above are the Electro-Eluter/Concentrator available from CBS Scientific, Del Mar, Calif. 92014; the preparative gel electrophoresis system available from Bethesda Research Laboratories, Bethesda, Md.; and the Trans-Blot Cell available from Bio-Rad, Richmond, Calif.
U.S. Pat. No. 4,787,963 to MacConnell discloses methods and means for hybridizing complementary nucleic acid molecules at an accelerated rate by electrophoretically moving unhybridized probe sequences successively in various directions along the surface of a nucleic acid impermeable membrane and in contact with the target sequences bound thereto. The rate of hybridization is increased due to an increase in the incidence of collisions between probe and target molecules.
MacConnell discloses performing electrophoretically-enhanced hybridization on a membrane assembly sandwiched between opposing solutions. It does not disclose a method of enhancing hybridization by applying electrophoretic force to a membrane within a gel.
U.S. Pat. No. 5,632,957 to Heller et al. discloses systems for performing molecular biological diagnoses, including nucleotide hybridization assays. The systems have a matrix of addressable microscopic locations on their surfaces, wherein each location is able to electronically control and direct the transport and attachment of specific binding entities (e.g., nucleic acids) to the locations, thereby increasing the rate and specificity of hybridization by concentrating hybridization reactants at specific microscopic locations. Any un-bound analytes or reactants can be removed by reversing the polarity of a micro-location.
Heller et al. at column 16, lines 16-18, discloses the use of "convective mass transport" as an alternative method to its preferred method of electrophoretic transport.
U.S. Pat. No. 5,310,650 to McMahon et al. discloses a method for assaying nucleotides on porous media, particularly microporous chromatographic media. The method increases the rate of hybridization by using capillary action to enhance interaction of probe and target.
None of the foregoing patents disclose centrifugation-enhanced and vacuum-enhanced hybridization methods. Moreover, it is unclear from the foregoing patents whether any of the methods are sensitive enough to distinguish a one-base mismatch from a perfect match between probe and target. Many applications require such sensitivity, particularly when a one-base mutation is all that distinguishes wild-type DNA and mutant-type DNA which is correlated with disease.
All references cited herein are incorporated herein by reference in their entireties.